Dantrolene is a synthetic hydantoin derivative used in the treatment of malignant hyperthermia, a rare, genetic sensitivity to volatile anesthetics. This drug acts by interaction with a regulator of intracellular Ca2+ release. The process of intracellular Ca2+ release is involved in many normal and pathophysiologic processes in skeletal muscle, heart and brain. The purpose of the present synthetic proposal is to create a nonexchangeable, tritiated derivative of dantrolene to be used in subcellular binding studies in order to pharmacologically characterize this putative receptor, and the synthesis of tritiated azidodantrolene, a photoactive congener, to be used in molecular identification of this regulator of cellular Ca2+. The synthesis of [3H]dantrolene and [3H]azidodantrolene begins with the reduction of the aryl methyl ether (containing either a nitro or an azido group at the para position of the phenyl ring) with Li(Et)3BT to give the corresponding alcohol. Oxidation with MnO2, followed by silica gel column purification, yields the corresponding aldehyde. Condensation with aminohydantoin in aqueous acetonitrile gives the acid form of these compounds. Reaction with sodium methoxide in methanol allows formation of the sodium salt. TITLE: Tritium Labelling of Azidodantrolene (Continued) We have recently characterized [3H]dantrolene (custom synthesized, 8.92 Ci/mmol) binding to subcellular membranes from skeletal muscle and found a single binding site by analysis of the specific binding curve. Recent evidence from another laboratory on the physiological action of the drug indicates that dantrolene's action is first activating, then inhibiting, intracellular Ca2+ release. This implies the presence of two binding sites. We hope to use the [3H] azido dantrolene synthesized in this project to a specific activity of >20 Ci/mmol to investigate the presence of a high affinity binding site in the nanomolar range. We will further use [3H]dantrolene to identify the molecule to which dantrolene binds and effects its inhibition of Ca2+ release, by a combination of photocrosslinking, SDS-polyacrylamide gel electrophoresis and autoradiography. Depending on the results achieved with the present compounds, we may need to increase our specific radioactivity even further. The results of the binding experiments with the tritium labelled azido-dantrolene may also require us to devise different methods of photocrosslinking in our experiments. In that case we will need to re-investigate the structure-activity relationships of newly synthesized congeners of dantrolene containing other photoreactive moieties. If they demonstrate biological activity, we will proceed to have them labelled at high specific radioactivity.